The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.

FOXP4 promotes proliferation of human spermatogonial stem cells / 亚洲男科学杂志(英文版)

Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.

Proliferation and differentiation of mouse spermatogonial stem cells on a three-dimensional surface composed of PCL/gel nanofibers / Proliferación y diferenciación de células madre espermatogónicas de ratón en una superficie tridimensional compuesta de nanofibras PCL / gel

Spermatogonial stem cells (SSCs) have self-renewal and differentiation capacity essential for sperm production throughout the male reproductive life. The electrospun polycaprolactone/gelatin (PCL/Gel) nanofibrous scaffold mimics important features of the extracellular matrix (ECM), which can provide a promising technique for the proliferation and differentiation of SSCs in vitro. The goal of the present study was to investigate the effects of PCL/Gel nanofibrous scaffold on the propagation and differentiation of neonate mouse SSCs (mSSCs). mSSCs were enzymatically isolated, and the cells were purified by differential plating method and seeded on scaffold. After 2 weeks, viability, colony number and diameter, and expression of specific spermatogonial cell genes were investigated. After mSSCs propagation, the cells were cultivated in a differentiation medium on the scaffold for another 2 weeks, and differentiating cells were analyzed by real-time PCR. The number of mSSC colony (P<0.01) and expression levels of specific spermatogonial genes Plzf and Inga6 (P<0.01) and also differentiation genes c-Kit, Tp1 and Ptm1 (P<0.05) were higher in scaffold group compared with control during the culture period. We conclude that mSSCs can be expanded and can differentiate toward spermatid cells on PCL/Gel nanofibrous scaffold with improved developmental parameters.

Las células madre espermatogónicas (CME) tienen capacidad de auto renovación y diferenciación esenciales para la producción de esperma a lo largo de la vida reproductiva masculina. El «scaffold¼ nanofibroso de policaprolactona / gelatina (PCL / Gel) electrohilado imita características importantes de la matriz extracelular (MEC), que puede proporcionar una técnica prometedora para la proliferación y diferenciación de CME in vitro. El objetivo del presente estudio fue investigar los efectos del «scaffold¼ nanofibroso PCL / Gel en la propagación y diferenciación de CME de ratones neonatos (mSSC). Los mSSC se aislaron enzimáticamente y las células se purificaron mediante un método de siembra diferencial y se sembraron en un «scaffold¼. Después de 2 semanas, se investigaron la viabilidad, el número y el diámetro de las colonias y la expresión de genes específicos de células espermatogónicas. Después de la propagación de mSSC, las células se cultivaron en un medio de diferenciación en el «scaffold¼ durante otras 2 semanas, y las células se analizaron mediante PCR en tiempo real. El número de colonias mSSC (P <0,01) y los niveles de expresión de los genes espermatogónicos específicos Plzf e Inga6 (P <0,01) y también los genes de diferenciación c-Kit, Tp1 y Ptm1 (P <0,05) fueron mayores en el grupo de «scaffold¼ en comparación con el control durante el período de cultivo. Concluimos que los mSSC pueden expandirse y diferenciarse en células espermátidas en un «scaffold¼ de nanofibras PCL / Gel con parámetros de desarrollo mejorados.

The Hsp60C gene in the 25F cytogenetic region in Drosophila melanogaster is essential for tracheal development and fertility.

Earlier studies have shown that of the four genes (Hsp60A, Hsp60B, Hsp60C, Hsp60D genes) predicted to encode the conserved Hsp60 family chaperones in Drosophila melanogaster, the Hsp60A gene (at the 10A polytene region) is expressed in all cell types of the organism and is essential from early embryonic stages, while the Hsp60B gene (at 21D region) is expressed only in testis, being essential for sperm individualization. In the present study, we characterized the Hsp60C gene (at 25F region), which shows high sequence homology with the other three Hsp60 genes of D. melanogaster. In situ hybridization of Hsp60C-specific riboprobe shows that expression of this gene begins in late embryonic stages (stage 14 onwards), particularly in the developing tracheal system and salivary glands; during larval and adult stages, it is widely expressed in many cell types but much more strongly in tracheae and in developing and differentiating germ cells. A P-insertion mutant (Hsp60C(1)) allele with the P transposon inserted at -251 position of the Hsp60C gene promoter was generated. This early larval recessive lethal mutation significantly reduces levels of Hsp60C transcripts in developing tracheae and this is associated with a variety of defects in the tracheal system, including lack of liquid clearance. About 10% of the homozygotes survive as weak, shortlived and completely sterile adults. Testes of the surviving mutant males are significantly smaller, with fewer spermatocytes, most of which do not develop beyond the round spermatid stage. In situ and Northern hybridizations show significantly reduced levels of the Hsp60C transcripts in Hsp60C(1) homozygous adult males. The absence of early meiotic stages in the Hsp60C(1) homozygous testes contrasts with the effect of testis-specific Hsp60B (21D) gene, whose mutation affects individualization of sperm bundles later in spermiogenesis. In view of the specific effects in tracheal development and in early stages of spermatogenesis, it is likely that, besides its functions as a chaperone, Hsp60C may have signalling functions and may also be involved in cation transport across the developing tracheal epithelial cells.

Histología testicular humana comparada, adulto joven y senil / Human testicular histology in young and senile men

La fisiología del envejecimiento implica cambios morfológicos que interfieren con la función testicular normal. El hombre senil mantiene la potencia fecundante, aunque su eficacia disminuye. Posiblemente, asociada a una presentación histopatológica testicular. Se utilizaron muestras de tejidos testicular de 3 individuos seniles (69 años), sometidos a orquiectomía terapéutica y de un joven adulto (25 años). Fallecido por causa desconocida. Las gónadas fueron procesadas por técnicas histológicas para Hematoxina-P.A.S. Al microscopio se evaluaron la morfometría, celularidad e histopatología del epitelio seminífero. En los resultados se obtuvo una reducción del diámetro tubular y altura del epitelio seminífero en los individuos seniles, al ser comparados con el individuo joven. Paralelamente, en los testículos de individuos seniles se describio una drástica disminución en el número de células de De Sertoli, y un poco menor para espermatogonias tipo A oscuras, A claras y B; con una relación gonia/Sertori alterada. Asociado a lo anterior, se encontró un porcentaje reducido de espermátidas redondas y alargadas y de espermatozoides, en lumen por túbulo. La evaluación histopatológica en los individuos seniles reveló un epitelio seminífero severamente dañado, con presencia de vacuolización, discontinuidad del epitelio y detención de la espermatogénesis. Por lo tanto, se concluye la existencia de un notorio efecto adverso del progreso de la edad hacia senil, en la estructura histológica testicular, involucrando las diferentes poblaciones celulares y la relación entre ellas, como también la integridad del epitelio seminífero